Transcriptional repression is emerging as an important feature of developmental processes. The Hairy/E(spl) family of bHLH proteins appear to function as dedicated repressors. The broad long term goal of proposed study is to determine the precise molecular mechanism(s) of Hairy action during Drosophila segmentation. This study will use genetic, developmental, molecular, and biochemical approaches to further characterize Hairy and two Hairy-interacting proteins to understand how they function as transcriptional repressors. The specific aims of this proposal are: (1) determine the structural and functional properties of dCtBP; (2) determine the structural and functional properties of h-NC13; and (3) investigation of the role of Hairy and its interacting proteins in Hairy-mediated transcriptional repression. The bHLH motif has been found in many genes required during diverse developmental processes such as segmentation, sex determination, neurogenesis, somitogenesis, and myogenesis in a wide range of organisms including Drosophila, vertebrates, yeast, plants, and nematodes. bHLH proteins are often involved in developmental switch decisions: they regulate the choice between alternate pathways and effect changes in cell fate. Mistakes in these regulatory steps often lead to developmental defects and the onset of cancers. Indeed, a functional relationship between bHLH proteins and oncology has also been demonstrated by the deregulated expression of the myc bHLHZip proto-oncogene in many cancers and the involvement of bHLH genes in acute T-cell leukemia. The numerous processes requiring bHLH proteins and the wealth of techniques/reagents available in Drosophila make in an excellent organism for studying the functions of HLH proteins in vivo. Results obtained from these projects are expected to have wide implications as Hairy/E(spl) proteins and mechanisms of transcriptional repression are conserved throughout organisms.